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Method for improving dianthus conversion rate based on tobacco rattle virus silencing system

A technology of tobacco crisp virus and dianthus, applied in biochemical equipment and methods, botany equipment and methods, genetic engineering, etc., can solve the problems of slow transformation effect and poor induction efficiency, and achieve fast effect, low cost, and detection high efficiency effect

Active Publication Date: 2018-10-26
HUAZHONG AGRI UNIV
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Problems solved by technology

Virus-induced gene silencing technology (virus-induced gene silence, VIGS) has been successfully applied to a variety of plants for rapid verification of gene function. Although previous reports have been made on the Dianthus VIGS system, the induction of Dianthus transformation after applying this system The efficiency is poor, only 20%, and the transformation effect is slow, and the albino phenotype does not appear until about 20 days after infection

Method used

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  • Method for improving dianthus conversion rate based on tobacco rattle virus silencing system
  • Method for improving dianthus conversion rate based on tobacco rattle virus silencing system
  • Method for improving dianthus conversion rate based on tobacco rattle virus silencing system

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Embodiment 1

[0045] (1) Cloning of DcPDS gene fragment

[0046] Take 0.5 g of fresh Dianthus leaves, extract the total RNA of the leaves with reference to the operation method of the Trizol RNA extraction kit manual of Treasure Bioengineering Dalian Co., Ltd., and perform reverse transcription with the M-MLV reverse transcription kit of Treasure Bioengineering Dalian Co., Ltd. to obtain For the cDNA of Dianthus leaves, according to the sequence information of the gene in the Dianthus library, the specific primer combination as described below was used to design the following specific primer combination to amplify the DcPDS gene fragment, and the specific sequence of the primer combination is as follows;

[0047] Forward primer DcPDS-F: 5'-CCATTGAAGGTGGTGTGTGTAGA-3' (sequence is the same as SEQ ID NO: 2)

[0048] Reverse primer DcPDS-R: 5'-TTGGGGTAAGCCCCCAAAGA-3' (sequence is the same as SEQ ID NO: 3)

[0049] The cDNA of Dianthus leaves was used as a template for PCR reaction. In a 20μL ...

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Abstract

The invention belongs to the technical field of gene engineering of decorative plants, and particularly relates to a method for improving dianthus conversion rate based on the tobacco rattle virus silencing system. The silencing system comprises a PDS gene sequence shown in SEQ ID NO: 1, a specific primer is designed according to the gene fragment of the dianthus, a 305bp cDNA fragment is amplified in the dianthus by PCR, the obtained fragment is connected to a TRV2 carrier, and a recombinant expression carrier TRV2-DcPDS is constructed. The dianthus is infected by using an agrobacterium-mediated mechanical damage method, and the gene function is identified by using the recombinant expression carrier TRV2-DcPDS to transform the dianthus by using a transient transformation method to inducesilencing of the gene of the dianthus. The method can be used for fast function identification and transgenic operation of the dianthus gene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of horticultural flowers, and in particular relates to a method for improving the conversion rate of carnation based on a tobacco crisp virus silencing system. The invention can be used to improve the conversion rate of carnation and quickly identify the function of endogenous genes of carnation. Background technique [0002] Dianthus is a perennial herbaceous plant of the family Caryophyllaceae, native to my country and East Asia. Dianthus has a long history of cultivation and has high ornamental value, garden use and economic value. It can be used in flower beds, flower borders, flower beds, potted plants, and can also be used as cut flowers. Dianthus can also absorb sulfur dioxide and chloride, and is an excellent air purification material; Dianthus is also an important scientific research model plant for studying the development of 5 cardinal flowers. Improving the ornamental valu...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/70A01H5/00A01H6/30C12Q1/6895
CPCC12N15/70C12N15/8203C12N15/8218C12Q1/6895
Inventor 傅小鹏林胜男包满珠张晓妮
Owner HUAZHONG AGRI UNIV
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